The present invention relates to the use of antigen binding proteins in a method of inhibiting the infectivity of viruses or other infectious agents, products and compositions comprising such proteins and methods for identifying and/or selecting antigen binding proteins capable of exhibiting such activity. In particular, the invention relates to a method for inhibiting viral infection using a monovalent antigen binding protein comprising a variable domain of a heavy chain derived from an immunoglobulin devoid of light chains, capable of binding to a virus.
Antibodies are protein molecules belonging to a group of immunoglobulins generated by the immune system in response to an antigen. The structure of most antibody molecules is based on a unit comprising four polypeptides, two identical heavy chains and two identical light chains, which are covalently linked together by disulphide bonds. Each of these chains is folded in discrete domains. The C-terminal regions of both heavy and light chains are conserved in sequence and are called the constant regions, comprising one or more so-called C-domains. The N-terminal regions of the heavy and light chains, also known as V-domains, are variable in sequence and determine the specificity of the antibody. The regions in the variable domains of the light and heavy chains (VL and VH respectively) responsible for antigen binding activity are known as the hypervariable or complementary determining regions (CDR).
Immunoglobulins capable of exhibiting the functional properties of the four-chain immunoglobulins described above but which comprise two heavy polypeptide chains and which furthermore are devoid of light polypeptide chains have been described (WO 94/04678, Casterman et al, 1994). Fragments corresponding to isolated VH domains (hereinafter VHH) are also disclosed. Methods for the preparation of such antibodies or fragments thereof on a large scale comprising transforming a mould or yeast with an expressible DNA sequence encoding the antibody or fragment are described in patent application WO 94/25591 (Unilever).
The immunoglobulins described in WO 94/04678, which may be isolated from the serum of Camelids, do not rely upon the association of heavy and light chain variable domains for the formation of the antigen-binding site but instead the heavy polypeptide chains alone naturally form the complete antigen binding site. These immunoglobulins, hereinafter referred to as xe2x80x9cheavy-chain immunoglobulinsxe2x80x9d are thus quite distinct from the heavy chains obtained by the degradation of common (four-chain) immunoglobulins or by direct cloning which contribute part only of the antigen-binding site and require a light chain partner for antigen-binding, thus forming a complete antigen binding site.
Antibodies or fragments thereof, have found application in a variety of uses where the specific nature of the antibody-antigen interaction can be used to advantage. These include such uses as diagnosis, therapy, immunoassays and purification processes. The use of antibodies, or fragments thereof, in inhibiting viral infection has received attention, for instance during active immunisation with inactivated virus preparations or viral antigens produced in recombinant cells or during passive immunisation by the administration of neutralising antibodies.
It has been reported in the literature that monovalent Fab antibody fragments can neutralise viruses. Cheung et al (1992), Journal of Virology, 66, 6714-6720, describe the production of the Fab domain of a rabies virus-neutralising antibody MAb-57 and further demonstrate that this monovalent fragment itself has virus-neutralising activity. Other publications also report the capability of human Fab monovalent antibody fragments to neutralise or inhibit viral activity (see for example, Williamson et al (1993), Proc. Natl. Acad. Sci. USA, 90, 4141-4145). Such methods are not suitable for wide scale industrial application as the cost of producing such classical antibody fragments renders the processes economically unfeasible.
An alternative approach to inhibiting viral replication using antibodies which has been described in the literature is to select antibodies to target enzymes produced by the virus. Martin et al, Protein Engineering, 10(5), 607-614 (1997) describes the use of a camelisedf VH antibody fragment to inhibit hepatitis C virus NS3 protease, thereby preventing cleavage of the viral poly-protein precursor.
Another industrial application in which economically viable solutions to the problem of viral infection are sought is the field of fermentation processing, particularly food processing.
Lactic acid bacteria: (LAB: Lactococci and Lactobacilli) play an important role in food fermentation processes such as the production of cheese or yoghurt. Often such fermentations are hampered by the sensitivity of the bacteria towards viruses, known as bacteriophage, which build up in these, often not aseptically performed, processes. A phage infection causes the LAB cells to lyse; during prolonged fermentations phage resistant cell populations can evolve, but this delay affects the production capacity severely, and the disturbed process yields a product of low quality. Sometimes the process has to be stopped prematurely, with complete loss of the batch of milk.
To date, the phage problem has mainly been approached by taking special precautions with respect to hygiene at the production facility, but this causes additional time delays. Another solution which has been proposed is the use of resistant LAB strains, but the regular appearance of adapted forms of bacteriophage forces the strains used to be changed from time to time in a procedure known as culture rotation. This has the disadvantage of requiring labour intensive monitoring of the production facilities and medium for the presence of phage and requires the availability of several sets of cultures with the same functional attributes, differing only in phage sensitivity. There therefore remains considerable commercial interest in the further development of methods for combating LAB phage infection.
One method, proposed by Geller et al (1998), J. Dairy Sci., 81, 895-900, involves the use of colostrum from cows immunised with lactococcal phage as a source of phage-neutralising (polyclonal) antibodies to prevent lytic infection of Lactococcus lactis in fermentations of phage-contaminated milk. This method does not provide a commercially viable solution to the problem, however. Not only is it extremely economically unattractive to produce antibodies in this way but furthermore, the addition of colostrum to milk does not have regulatory approval.
An alternative approach, which makes use of multivalent, multispecific antigen binding proteins comprising a polypeptide comprising in series two or more single domain binding units, preferably variable domains of a heavy chain derived from an immunoglobulin naturally devoid of light chains, to reduce the infectivity of LAB phages by cross-linking or agglutination is exemplified in the Applicant""s co-pending patent application number PCT/EP98/06991, filed Oct. 26, 1998.
There remains a continuing need for the development of improved methods of inhibiting or neutralising viral infection. In particular, there remains continuing interest in development of methods which can be applied economically on a scale appropriate for industrial use.
Accordingly, the invention provides in one aspect a method of inhibiting viral infection using a monovalent antigen binding protein comprising a single variable domain binding unit, or a functional equivalent thereof, capable of binding to a virus.
In another aspect the invention provides the use of a monovalent antigen binding protein comprising a single variable domain binding unit or a functional equivalent thereof capable of binding to a virus in inhibiting viral infection.
The invention also provides the use of a monovalent antigen binding protein comprising a single variable domain binding unit or a functional equivalent thereof capable of binding to a virus in the preparation of a medicament for inhibiting viral infection.
Also provided are monovalent antigen binding proteins comprising a single variable domain binding unit capable of binding to a virus, nucleotide sequences encoding such proteins, cloning and expression vectors comprising such nucleotide sequences, host cells transformed with vectors comprising such nucleotide sequences, and food, cosmetic and pharmaceutical products comprising such proteins.
In a further aspect, the invention provides a method for selecting an antigen binding protein capable of inhibiting viral infection of a host cell comprising the steps of:
i) complexing an antigen binding protein with a target virus,
ii) exposing the antigen binding protein-virus complex of step (i) to an excess of host cells,
iii) removing the host cells and any associated antigen binding protein-virus complex,
iv) capturing antigen binding protein-virus complex not taken up by the host cells in step (ii) with virus specific ligands to separate virus specific antigen binding proteins from non-binding proteins.
The invention also provides a method for identifying an antigen binding protein capable of inhibiting bacteriophage infection of a lactic acid bacterial cell host comprising the steps of:
i) culturing of bacterial host cells in the presence of antigen binding protein and bacteriophage,
ii) assaying said culture for active cell growth manifest in a change in pH of the culture growth medium.
As used herein, a single variable domain binding unit means an immunoglobulin variable domain or a functional equivalent thereof which forms a complete antigen binding site. This may be derived from natural sources or synthetically produced. The terms xe2x80x98immunoglobulinxe2x80x99 and xe2x80x98antibodyxe2x80x99 are used synonymously throughout the specification, unless indicated otherwise.
A xe2x80x98functional equivalentxe2x80x99 of an imunoglobulin variable domain is any homogolous protein molecule which has similar binding specificity. A functional equivalent may be characterised by an insertion, deletion or substitution of one or more amino acid residues in the sequence of the immunoglobulin variable domain. Suitably, the amino acid sequence of the functional equivalent has at least 60% similarity, preferably at least 80%, more preferably at least 90% similarity to the amino acid sequence of the immunoglobulin variable domain.
Inhibition of viral infection includes but is not limited to inhibition of infection by blocking essential sites on the viral particle, such as the receptor binding protein of the virus by which the virus attaches to the host cell during the first step of infection. Inhibition may be total or partial. The terms xe2x80x98inhibitxe2x80x99 and xe2x80x98neutralisexe2x80x99 are used synonomously herein.
The term xe2x80x98virusxe2x80x99 includes within its scope viruses, which infect bacterial host cells, known as bacteriophages. Binding to a virus includes binding to one or more molecules located at the surface of the virus particle.